Protein conferring an inducible resistance to glycopeptides, particularly in gram-positive bacteria

ABSTRACT

The invention relates to a protein VanB involved, in Gram-positive bacteria, in resistance to glycopeptides, particularly to vancomycine, said resistance being of the type inducible by the vancomycine and non-inducible by teicoplanine. The invention also relates to the utilisation of fragments of nucleotides of the gene van B for the detection of resistances to glycopeptides.

The invention relates to the polypeptides associated with the expressionof a resistance to antibiotics of the glycopeptide family, thisresistance being of a type inducible by vancomycin and not inducible byteicoplanin, particular in the Gram-positive bacteria, in particular inthe family of the Gram-positive cocci. The invention also relates to anucleotide sequence coding for these polypeptides. It also relates tothe use of these polypeptides and their nucleotide sequence as agentsfor the in vitro detection of resistance to glycopeptides. Among theGram-positive cocci, the invention relates more particularly to theenterococci, the streptococci and the staphylococci.

The glycopeptides, which include vancomycin and teicoplanin, areantibiotic inhibitors of the synthesis of the bacterial cell wall. Theseantibiotics are very much used for the treatment of severe infectionsdue to Gram-positive cocci (enterococci, streptococci and staphylococci)in particular in cases of allergy and resistance to the penicillins.

Up to 1986 vancomycin proved to be efficacious against almost allstrains of enterococci.

The activity of the glycopeptides depends on the formation of a complexbetween the antibiotic and the peptidoglycan precursors more than ontheir direct interaction with enzymes of cell wall metabolism. Inparticular, it has been observed that the glycopeptides bind to theterminal D-alanyl-D-alanine (D-ala-D-ala) residues of the peptidoglycanprecursors.

Several phenotypes of resistance to the glycopeptides have beendemonstrated; in particular, strains resistant to a high level ofglycopeptides and strains resistant to low concentration levels.

By strain resistant to a high level is meant a strain of bacteria, inparticular a strain of Gram-positive cocci, for which the minimalinhibitory concentrations (MIC) of vancomycin and teicoplanin are higherthan 32 and 8 μg/ml, respectively. The MIC of vancomycin towards strainswith low-level resistance are included between 8 and 32 μg/ml. The VanBphenotype is characterized by a resistance inducible by vancomycin butnot inducible by teicoplanin. Once induced, this resistance may existagainst different glycopeptides, in particular against vancomycin and/orteicoplanin, and at variable levels.

The strains of enterococci corresponding to the VanB phenotype (class B)are in particular strains of E. faecalis and E faecium.

Al-Obed S et al. (FEMS Microbiology Letters 70 (1990) 101-106) have thuscompared the resistance proteins to glycopeptides, inducible byvancomycin, in four strains of Enterococci, and have deduced from theircomparison the existence of three types of proteins, one of these typesbeing present in the E. faecium strain resistant to low levels ofvancomycin. According to the authors of this publication, a protein ofmolecular weight of about 39.5 kDa is induced in the strains withlow-level resistance and this resistance is linked to induction byvancomycin. These strains were also reported to exhibit a resistance toteicoplanin, also induced by vancomycin.

According to Al-Obeid et al., this protein of 39.5 kDa is present inmultiple forms but the nature of this multiplicity has not been studied.According to these authors there might exist a structural specificitydepending an the species of bacteria concerned and the level Ofresistance, which needs to be confirmed.

In this publication Al-Obeid et al. described 11 amino acids of theN-terminal sequence of the protein of 39.5 kDa and observed that thissequence exhibited about 70% homology with many membrane proteins ofprokaryotic or eukaryotic origin having diverse functions. According tothe authors this comparison did not allow the possible function of theprotein to be established. Finally, Al-Obeid et al. noticed that otherproteins are induced, although to a lesser degree.

The invention relates to peptides, polypeptides or proteins implicatedin the expression of a resistance to antibiotics of the glycopeptidefamily and in particular to vancomycin and/or teicoplanin as well asnucleotide sequences coding for such polypeptides. The resistance inquestion above is of a type inducible by vancomycin but not byteicoplanin.

The expressions "implicated in the expression of a resistance" or"implicated in a resistance" signify that the protein of the inventionis necessary in order for the resistance to be manifest.

The invention also relates to nucleotide probes utilizable for thedetection of a resistance to the glycopeptides, in particular by meansof the polymerase chain reaction (PCR), or y assays involvingantibodies.

Thus, the object of the invention is a VanB protein characterized inthat it comprises the amino acid sequence I (SEQ. ID NO:2), and in thatit participates in the resistance to glycopeptides, in particular tovancomycin, this resistance being of a type inducible by vancomycin andnot by teicoplanin in Gram-positive bacteria

By the expression "inducible resistance" is meant the capacity of aspecific Gram-positive bacterium, in particular of a specificEnterococcus strain, to produce a VanB protein in the presence of aconcentration of 0.05 to 1 μl/ml of vancomycin.

The resistance to one or more defined glycopeptides may result in thepersistence of an infection due to microbes usually sensitive to theglycopeptides, or may be detected by means of an antibiogram(particularly for high levels of resistance), the MIC, hybridizationwith probes (after amplification by the PCR, for example).

According to a first embodiment of the invention, the VanB protein ischaracterized in that it is implicated in an inducible resistance toglycopeptides, and in particular to vancomycin, in enterococci and forexample in strains of the genus E. faecium or E. faecalis.

The invention also relates to a VanB protein characterized in that itcomprises an amino acid sequence modified with respect to sequence I bydeletion, insertion, or replacement of one or more amino acids, providedthat the VanB protein thus modified is implicated in Gram-positivebacteria in a resistance to glycopeptides, in particular to vancomycin,this resistance being of a type inducible by vancomycin, but notinducible by teicoplanin

Also included in the framework of the invention is any peptide fragmentof the VanB protein characterized in that it corresponds to the aminoacid sequence I or any part of this sequence functionally associatedwith the inducible resistance to glycopeptides, in particular tovancomycin, in Gram-positive bacteria, for example bacteria of thefamily of the enterococci.

Advantageously peptide fragments of the invention exhibit additionallyor alternatively antigenic properties and are hence recognized byantibodies formed against the VanB protein.

A particular fragment of sequence I corresponds for example to thesequence of residues 110-305 of SEQ. ID NO:2 or includes the sequence ofresidues 110-305 of SEQ. ID NO:2.

According to another embodiment of the invention, these antigens arespecific for the VanB protein and thus not recognized by antibodiesrecognizing the VanA and VanC proteins such as described in the patentapplication EP 91920753.

In addition the invention relates to a nucleotide sequence characterizedin that it codes for a VanB protein implicated in resistance toglycopeptides, in particular to vancomycin, in Gram-positive bacteria,this resistance being of a type inducible by vancomycin but notinducible by teicoplanin, said VanB protein comprising the amino acidsequence I or in that it is a DNA sequence complementary to this codingsequence or a corresponding RNA sequence.

By complementary sequence is meant any DNA sequence whose nucleotidesare complementary to those of sequence I and whose orientation isreversed.

A particular nucleotide sequence corresponding to this definition ischaracterized in that it comprises the following nucleotide sequence IIor a nucleotide sequence modified with respect to II provided that itcodes for a protein implicated in resistance to glycopeptides, inparticular to vancomycin, in Gram-positive bacteria, this resistancebeing of a type inducible by vancomycin but not inducible byteicoplanin. SEQ. ID NO:1)GAGCGTGTGCTGCGAGATACCACAGAAAACAATCAGAATTGTCTTAACTTTGAAAGGAGTTTACAGCATGAATAAAATAAAAGTCGCAATTATCTTCGGCGGTTGCTCGGAGGAACATGATGTGTCGGTAAAATCCGCAATAGAAATTGCTGCGAACATTAATACTGAAAAATTCGATCCGCACTACATCGGAATTACAAAAAACGGCGTATGGAAGCTATGCAAGAAGCCATGTACGGAATGGGAAGCCGATAGTCTCCCCGCCATATTCTCCCCGGATAGGAAAACGCATGGTCTGCTTGTCATGAAAGAAAGAGAATACGAAACTCGGCGTATTGACGTGGCTTTCCCGGTTTTGCATGGCAAATGCGGGGAGGATGGTGCGATACAGGGTCTGTTTGAATTGTCTGGTATCCCCTATGTAGGCTGCGATATTCAAAGCTCCGCAGCTTGCATGGACAAATCACTGGCCTACATTCTTACAAAAAATGCGGGCATCGCCGTCCCCGAATTTCAAATGATTGAAAAAGGTGACAAACCGGAGGCGAGGACGCTTACCTACCCTGTCTTTGTGAAGCCGGCACGGTCAGGTTCGTCCTTTGGCGTAACCAAAGTAAACAGTACGGAAGAACTAAACGCTGCGATAGAAGCAGCAGGACAATATGATGGAAAAATCTTAATTGAGCAAGCGATTTCOGGCTGTGAGGTCGGCTGCGCGGTCATGGGAAACGAGGATGATTTGATTGTCGGCGAAGTGGATCAAATCCGGTTGAGCCACGGTATCTTCCGCATCCATCAGGAAAACGAGCCGGAAAAAGGCTCAGAGAATGCGATGATTATCGTTCCAGCAGACATTCCGGTCGAGGAACGAAATCGGGTGCAAGAAACGGCAAAGAAAGTATATCGGGTGCTTGGATGCAGAGGGCTTGCTCGTGTTGATCTTTTTTTGCAGGAGGATGGCGGCATCGTTCTAAACGAGGTCCAATACCCTGCCCGGTTTTACATCGTACAGCCGCTATCCACGCATGGCGGCTGCCGCAGGAATCACGCTTCCCGCACTAATTGACAGCCTGATTACATTGGCGATAGAGAGGTGACCCGTATGGAAAATGGTTTTTTGTTTTTTAGATGAAATGTTGCA

Generally speaking the sect of the invention is also a nucleotidefragment characterized in that it is capable of hybridizing understringent conditions with a sequence such as defined in sequence IIabove,

The stringent conditions are the following:

reaction temperature of 65° C. overnight in a solution containing 0.1%SDS, 0.7% skimmed milk powder, 6×SSC (1×SSC=0.15M NaCl and 0.015M sodiumcitrate at pH=7.0) washes at room temperature in 2×SSC-0.1% SDS, then at65° C. in 0.2 SSC-0.1% SDS.

Advantageously a nucleotide fragment corresponding to the previousdefinition will have at least 15 nucleotides, and preferably at least20.

For this purpose a particular nucleotide sequence comprises thefollowing sequence: (SEQ. ID NO:3)

TCTGTTTGAATTGTCTGGTATCCCCTATGTAGGCTGCGATATTCAkzGCTCCGCAGCTTGCATGGACAAATCACTGGCCTACATTCTTACAAAAAATGCGGGCATCGCCGTCCCCGAATTTCAAATGATTGAAAAAGGTGACAAACCGGAGGCGAGGACGCTTACCTACCCTGTCTTTGTGAAGCCGGCACGGTCAGGTTCGTCCTTTGGCGTAACCAAAGTAAACAGTACGGAAGAACTAAACGCTGCGATAGAAGCAGCAGGACAATATGATGGAAAAATCTTAATTGAGCAAGCGATTTCGGGCTGTGAGGTCGGCTGCGCGGTCATGGGAAACGAGGATGATTTGATTGTCGGCGAAGTGGATCAAATCCGGTTGAGCCACGGTATCTTCCGCATCCATCAGGAAAACGAGCCGGAAAAAGGCTCAGAGAATGCGATGATTATCGTTCCAGCAGACATTCCGGTCGAGGAACGAAATCGGGTGCAAGAAACGGCAAAGAAAGTATATCGGGTGCTTGGATGCAGAGGGCTTGCTCGTGTTGATCTTTTTTTGCAGGAGGATGGCGGCATCGTTCTAAACGAGGTC

The peptides and polypeptides of the invention make it possible todefine a genotypic class, characterized by the capacity of thenucleotide sequences coding for these peptides to hybridize understringent conditions with the sequance II constituting a probe.

These fragments may be used as primers for carrying out amplificationreactions, or as probes.

Particularly valuable probes correspond to the following sequences:(SEQ. ID NO:4-5)

primer 1:5' ATGGGAAGCCGATAGTC 3' (SEQ. ID NO:4) (positions 241-258 ofnucleotides of sequence I)

primer 2:5' GATTTCGTTCCTCGACC 3' (SEQ ID NO:5) (complementary reversesequence of the nucleotide fragment 860-877 of sequence I).

Nucleotide probes according to the invention may be specific for thedetection in Gram-positive bacteria of sequences coding for a VanBprotein implicated in the resistance to glycopeptides, in particular tovancomycin and/or teicoplanin, this resistance being inducible inconformity with the previous definition, these probes being in additionuniversal among these sequences.

By probes specific for VanB is meant any oligonucleotide hybridizingwith a nucleotide sequence coding for a VanB protein according to theinvention as described in the preceding pages, and not exhibitingcross-hybridization or amplification (PCR) reactions with sequencespresent in all of the sensitive strains.

A particular nucleotide fragment according to the invention ischaracterized in that it does not hybridize under stringent conditionswith the DNA of strains of enterococci sensitive to vancomycin, inparticular with the DNA of the strains E. faecalis JH2--2 and E. faeciumBM4107.

These reference strains have been described by Jacobs and Hobbs (J.Bacteriol. 117, 1974, 360-372) and Leclercq et al. (Antimicrob. AgentsChemother. 33, 1989), respectively.

Another useful nucleotide fragment in the framework of the invention isspecific for the vanB gene to the extent that it does not hybridizeunder stringent conditions with the vanA and vanC genes as described inthe PCT application 91920753.

A particularly useful nucleotide fragment in the framework of theinvention is the fragment corresponding to sequence II.

This fragment is an internal fragment derived frcm the gene implicatedin the resistance to strains of enterococci. The resistance may exist atvariable concentration levels of glycopeptides

The invention also relates to nucleotide fragments modified with respectto the foregoing by mutation, addition or deletion of nucleotides,provided that the fragment thus modified either codes for a fragment ofthe functional VanB protein as regards its property of the resistance toglycopeptides, in particular to vancomycin, under conditions describedabove, or hybridizes with the vanB gene.

Should the nucleotide fragments be used as probes, labelling isperformed by the standard techniques. As examples, radioactive orenzymatic markers should be used.

Nucleotide fragments according to the invention may be used as primersto carry out the amplification of the nucleic acid contained in a givenbiological sample, for example by PCR

Moreover, the invention relates to a recombinant DNA sequencecharacterized in that it comprises a nucleotide sequence described aboveunder the control of regulatory elements likely to be involved in thecloning and expression of a gene implicated in a resistance, of a typeinducible by vancomycin and not inducible by teicoplanin, to antibioticsof the glycopeptide family, in particular vancomycin, in a defined host.

This gene implicated in the resistance is for example the vanB genewhich comprises the nucleotide sequence II or any functional part interms of inducible resistance derived from a sequence hybridizing withsequence II.

The invention also relates to a recombinant vector for the cloning andexpression, characterized in that it comprises a nucleotide sequencedescribed above at a site inessential for its replication, optionallyunder the control of regulatory elements likely to be involved in theexpression of a resistance, of a type inducible by vancomycin and not byteicoplanin, to antibiotics of the glycopeptide family, in particularvancomycin, in a defined host.

Particular vectors are for example plasmids, phages, cosmids, YACs.

A preferred vector is the plasmid pAT201 deposited with the C.N.C.M. on11 Dec. 1992 under the number I-1277.

Another preferred vector is the plasmid pAT202 formed from the plasmidpUC19Ω containing a 3.3 kb fragment containing the vanB gene ofEnterococcus faecalis V583 (HindIII/KpnI).

pAT202 was introduced into E. coli JM83 and deposited with the C.N.C.M.on 29 Mar. 1993 under the number I-1291 (identification E. coli BM2973).

These vectors may be used to transform or transfect cell hosts in orderto clone or express the nucleotide sequences of the invention.

A recombinant cell host according to the invention is characterized inthat it is modified by a nucleotide sequence or a vector describedabove.

The cell host is preferably modified by this sequence under conditionspermitting the expression of a functional VanB protein as regardsinducible resistance to glycopeptides.

The object of the invention is also a recombinant VanB protein such asobtained from a recombinant cell host according to the previousdefinition, the VanB protein obtained being characterized in that itspeptide skeleton comprises the above amino acid sequence, and in that itis implicated in a resistance to glycopeptides, in particular tovancomycin, in Gram-positive bacteria, this resistance being of a typeinducible by vancomycin but not inducible by teicoplanin.

The VanB protein according to the invention makes it possible to preparemonoclonal or polyclonal antibodies characterized in that they recognizespecifically the VanB protein or a peptide fragment described above.

These antibodies may be obtained according to the standard methods forthe production of antibodies. In particular for the preparation of themonoclonal antibodies recourse should be had to the method of Kohler andMilstein according to which monoclonal antibodies are prepared by cellfusion between myeloma cells and spleen cells of mice previouslyimmunized with a polypeptide or a composition according to theinvention, in conformity with the standard procedure

The antibodies of the invention can advantageously be used for thedetection of the presence of proteins characteristic of a resistance tothe glycopeptides, in particular to vancomycin and teicoplanin, thisresistance being of the type inducible by vancomycin but not inducibleby teicoplanin.

Also included in the framework of the invention is a kit for the invitro diagnosis in a biological sample of the presence of strainsresistant to glycopeptides after induction, in particular by vancomycinbut not by teicoplanin, these strains belonging in particular to theGram-positive cocci, in particular in that they are strains ofenterococci, for example E. faecium, characterized in that it contains:

optionally labelled antibodies described above,

a reagent for the detection of an immunological reaction of theantigen-antibody type,

optionally, reagents for lysing the cells of the tested sample,

optionally, a defined concentration of vancomycin to induce resistance.

The invention also relates to a kit such as that defined above whichcontains in addition antibodies specifically directed against the VanAprotein and/or antibodies specifically directed against the VanCprotein.

According to another embodiment of the invention, the kit enablesresistance corresponding to a phenotype VanA VanB or VanC to be detectedindiscriminately and contains antibodies recognizing VanA, VanB and VanCThese antibodies may be selected by their capacity to recognize anepitope common to the three proteins. It may also be a mixture ofantibodies recognizing different epitopes, specific to each cf theproteins.

According to another embodiment of the invention, a kit for the in vitrodiagnosis of the presence of strains resistant to low levels ofglycopeptides, resistant in particular to vancomycin, is characterizedin that it contains:

a nucleotide probe capable of hybridizing under stringent conditionswith a nucleotide sequence of the vanB gene, and optionally,

nucleoside triphosphates dATP, dCTP, dTTP, dGTP,

a DNA plymerase.

Another detection kit contains in addition nucleotides capable ofhybridizing specifically with the vanA gene and a probe capable ofhybridizing specifically with the vanC gene.

This kit may be advantageously used for the detection of a resistance inGram-positive cocci, in particular in enterococci, for example in E.faecium.

The invention also relates to a kit for the in vitro detection of aresistance to glycopeptides, in particular to vancomycin, thisresistance corresponding to one of the phenotypes VanA, VanB or VanC,the kit containing:

a nucleotide probe hybridizing with the genes vanA, vanB and vanC,

nucleoside triphosphates dATP, dCTP, dTTP and dGTP,

a DNA plymerase.

The invention also relates to a procedure for the in vitro detection ofthe presence of strains resistant to glycopeptide in particular tovancomycin and/cr teicoplanin, these strains belonging in particular tothe family of the Gram-positive cocci, in particular in that they arestrains of enterococci, for example E faecium or E. faecalis,characterized in that it comprises:

a) the placing of a biological sample likely to contain the resistantstrains in contact with a primer constituted by a nucleotide fragmentaccording to the invention such as that described above, capable ofhybridizing with the nucleotide sequence under investigation andimplicated in the expression of the resistance, this sequence being usedas matrix in the presence of the 4 different nucleoside phosphates and apolymerase under conditions of hybridization such that for eachnucleotide sequence having hybridized with a primer, an elongationproduct of each primer complementary to the matrix is synthesized,

b) the separation of the matrix from the elongation product obtained,this latter being then also able to behave as a matrix,

c) repetition of step a) so as to obtain a detectable quantity of thenucleotide sequences investigated,

d) the detection of the amplification product of the nucleotidesequences.

The probe used may thus be specific for the nucleotide sequence II or asequence hybridizing with sequence II under stringent conditions. Underthese conditions, the procedure according to the invention makespossible the detection of a resistance to glycopeptides, this resistancebeing inducible by vancomycin but not inducible by teicoplanin.

According to a particular embodiment of the invention, this procedurealso comprises the placing of the biological sample in contact with aspecific nucleotide fragment of the vanA gene and/or a specificnucleotide fragment of the vanC gene. In this case the procedureaccording to the invention advantageously makes possible the detectionof different phenotypes of resistance.

According to another embodiment, a resistance corresponding to aphenotype VanA, VanB Cr VanC will de detected indiscriminately by usinga probe common to the genes vanA, vanB or vanC. Such a probe may beconstructed from the aligned polypeptide sequences of FIG. 2.

Other characteristics and advantages of the invention will becomeapparent in the following Examples and Figures:

FIGURES FIG. 1

Nucleotide and amino acid sequences corresponding to the vanB gene (SEQ.ID NO:1-2). The nucleotide sequences of the two strands was determinedfrom the insert contained in pUC18 by the dideoxy chain terminationmethod (Sanger et al., 1977, Proc. Natl. Acad. Sci. USA, 74: 5463-5467)using T7 DNA polymerase. The RBS sequence underlined represents theShine-Dalgarno sequence for ribosome binding.

FIG. 2

Alignment of the deduced amino aid sequence of the VanB protein andcorresponding regions of VanA, VanC, DdLA and DdLB of E. coli(Dutka-Malen et al., 1992 Gene, 112: 53-58), Ddl of E. faecalis V583 andDdlA of S. typhimurium (Daub et al., Biochemistry 27 1988 3701-3708)(SEQ. ID NO:2, 6-11). The identical amino acids (I) and the conservativesubstitutions (C) in the 7 sequences have been indicated beneath thealignment. In order to permit the classification of conservativesubstitutions, the amino acids have been regrouped as follows: RK,LFPMVI, STQNC, AGW, H, ED and Y The domains 1, 2, 3 and 4 correspond toregions of high homology.

FIG. 3

Oligonucleotides V1 and V2 used to amplify the DNA of the vanB gene(SEQ. ID NO:12-15).

FIG. 4

Nucleotide sequence of the ddl gene of E. faecalis V583 and thecorresponding amino acid sequence (SEQ. ID NO:16-17). The plasmid pAT203was constructed by subcloning the DNA of λ recombinant bacteriophagepartially digested with Sau3AI (Pharmacia) in pUC19 digested with BamHI(Pharmacia). The 15 kb insert of pAT203 contains the ddl gene. Thenucleotide sequence of 1079 consecutive bp of pAT203 was determined onboth strands by the dideoxy chain termination method. The first basepair of the sequence is defined as position 1. The ribosome bindingsequence RBS is at position 19 upstream from the start codon TTG Thestop codon TTA is indicated. The deduced amino acid sequence of the Ddlprotein is shown.

EXPERIMENTAL APPROACH

The antibiotics of the glycopeptide family such as vancomycin (Vm) andteicoplanin (Te) bind to the C-terminal D-Ala residues of thepeptidoglycan precursors thus blocking their incorporation into thebacterial cell wall (Reynolds, P.E. 1989 Eur. J. Qin. Microb Infect.Dis. 8: 943-950). The D-Ala residues are incorporated into theprecursors of the cell wall in the form of dipeptides synthesized byD-Ala:D-ala ligases (DDL) (Walsh, C.T. 1989 J. Biol. Chem. 264:2393-2396). The VanA ligase synthesizes the dipeptide D-Ala-D-lac whichsubstitutes for D-Ala-D-Ala leading to the synthesis of precursors whichbind vancomycin with reduced affinity (Bugg et al., Biochemistry 30:10408-10415 (1991), Handwerger et al., J. Bacteriol. 174: 5982-5984(1992), Messer et Reynolds, FEMS Microbid. Letters 94: 195-200 (1992)).

The resistance to the glycopeptides in the enterococci is heterogeneous(Dutka-Male et al., 1990 Antimicrobiol Agents Chemother. 34: 1875-1879).

The resistance proteins VanA and VanC (see patent application EP91920753.0 of 29 Oct. 1991) show a 30 to 37% homology (the details aregiven in Table III) with the amino acids of the D-Ala: D-Ala ligases(Ala=alanine) of E. coli (Dutka-Malen et al., 1992 Gene 112: 53-58). Thestructural genes for the VanA and VanC proteins do not hybridize withthe DNA of the strains with the VanB phenotype (Dutka-Malen et al.,1990, Leclercq et al., Antimicrob Agents Chemother. 36: 2005-2008(1992)).

The inventors have succeeded in identifying the nucleotide sequenceimplicated in the properties of resistance to vancnycin of strains ofenterococci having the VanB phenotype and resistant after induction withvancomycin.

Bacterial strains: 39 isolates of E. faecium (28 strains) and E faecalis(11 strains) resistant to low and high concentrations of vancomycin andsensitive to teicoplanin were studied (Table II). Among these strains 24isolates including E. faecalis V583 (Sahm D. Et al., Antimicrob. AgentsChemother. 1989, 33: 1588-91) and E faecium D366 (Gutmann L. Et al.,Antimicrob. Agents Chemother. 1992, 36: 77-80) were resistant to lowconcentrations of vancomycin on the basis of a disk sensitivity test.These strains belong to the class B phenotype. 15 isolates resistant tohigh concentrations of vancomycin (MIC ≧128 μg/ml) including E. faecalisstrain V583-2 (Zarlenga LJ. Et al., Antimicrob. Agents Chemother. 1992,36: 902-5), which is a spontaneous mutant of V553 as well as UMH-1(Schwalbe R. Et al., Abstract A-117, in Abstracts of the 91st GeneralMeeting of the American Society for Microbiology, Dallas, Tex. AmericanSociety for Microbiology, 1991) were also studied. The control strainswere well-characterized strains of enterococci belonging to thephenotypes A and C and hybridizing with the probes VanA and VanC,respectively. In particular there are 6 clinical isolates of E. faeciumhighly resistant to vancomycin and to teicoplanin, including BM4147. Thestrains of E. gallinarum including BM4147 belonging to class C were alsoused as controls. Strains of E. casseliflavus are also used as controls,including the strain ATCC 25788, which are isolates intrinsicallyresistant to low levels of vancomycin and sensitive to teicoplanin(Leclercq R et al., Antimicrob. Agents Chemother. 1992, 36: 2005-8).

The following strains were also studied: Erysipelothrix rhusiopathiaeA124 (Institute Pasteur collection), Lactobacillus brevis ATCC 14869,Lactobacillus casei ATCC 393, Lactobacillus confusus ATCC 10881,Lactobacillus fermentum ATCC 9338, Lactobacillus plantarum ATCC 8014,Lactobacillus reuteri ATCC 23272, Lactobacillus rhamnosus ATCC 7469,Lactobacillus salivarius ATCC 11741, Pediococcus acidilacti ATCC 8042,Pediococcus pentosaceus ATCC 33316, and Leuconostoc mesenteroides CIP16407. The following enterococci sensitive to antibiotics of theglycopeptide family are used as negative controls: E. durans ATCC 19432,E. faecium ATCC 19434, BM4107 (Leclercq R et al., Antimicrob. AgentsChemother. 1992, 36: 2005-8), and MT10R (Gutmann L et al., Antimicrob.Agents Chemother., 1992, 36: 77-80), strain sensitive to vancomycinderived from D366; E. faecalis ATCC 29212, ATCC 33186, JH2-2 (Leclercq Ret al., Antimicrob. Agents Chemother. 1992, 36: 2005-8) and V583-C1,strain sensitive to vancomycin derived from V583 (Table II) and aclinical isolate of E. faecium and E. faecalis. Characteristics ofreference strains are depicted in Table I. E. faecium BM4107 and E.faecalis JH2-2, resistant to both rifampin and fusidic acid (Leclercq Ret al., Antimicrob. Agents Chemother. 1992, 36: 2005-8) were used asreceptor strains for conjugation experiments.

Identification of the enterococci

The enterococci were identified by the method of Facklam and Collins, J.Qin. Microbiol. 1989, 27: 731-4). The identification of the species wasbased on the tests of potassium tellurite reduction and the productionof acids from carbohydrates on bands of API 20 streptococci (bioMerieux,Marcy l' Etoile France). The tests of mobility at 30° C. andfermentation of carbohydrates were used to distinguish E. gallinarum andE. Casseliflavus from E. faecium and E. faecalis. The strains of E.Casseliflavus were distinguished from the strains of E. gallinarum onthe basis of the production of a yellow pigment on the agar.

Medium

A brain-heart medium and agar (Difco Laboratories, Detroit, Mich.) wereused. Sensitivity tests were performed an Mueller-Hinton agar(Diagnostics Pasteur, Marne LaCoquette, France). All of the incubationswere performed at 37° C.

Determination of the in vitro sensitivity to the antibiotics.

The disk diffusion test with disks containing 30 μg of vancomycin or 30μg of teicoplanin (Diagnostics Pasteur) was used for the initialscreening. The method of Steers et al. with 10⁴ CFU per spot was used todetermine the MIC of the antibiotics (Steers E. Et al., Antibiot.Chemother. (Basel) 1959, 9: 307-11)

Transfer of the character of resistance to an antibiotic

The conjugation on filters was carried out according to the proceduredescribed by Dutka-Malen S. Et al., Antimicrob. Agents Chemother. 1990,34: 1875-9. The antibiotic concentrations for the selection of thetransconjugates were the following: rifampin: 20 μg/ml; fusidic acid:10μg/ml and vancomycin: 4 and 8 μg/ml.

Enzymes and reagents

Lysozyme was obtained from the Sigma Chemical Co.(St. Louis, Mo.). RNaseA (bovine pancreas) and proteinase K were obtained from Calbiochem.Co.(San Diego, Calif.). {∝-32p} dCTP and the triethylammonium salt(specific activity 3000 CI/mmol) were obtained from the RadiochemicalCenter, Amershan, Great Britain. Teicoplanin was obtained from GruppoLepetit (Milan, Italy) and vancomycin was obtained from Eli Lilly & Co(Indianapolis, Ind.).

The oligonucleotides V1 and V2 described in the patent application EP91920753.0 made possible the amplification by means of the PCR techniqueof fragments internal to the genes coding for the proteins VanA, VanCand D-Ala: D-Ala ligases (Dutka-Malen et al., 1992 Gene 112: 53-58).

The amplification of the vanB gene was carried out with theoligonucleotides V1 and V2 and the DNA (20 ng) of Enterococcus faecalisV583 (Sahm et al., 1989 Antimicrob. Agents Chemother.33: 1588-1591).

To carry out this amplification the technique described in thepublication of Dutka-Malen et al., 1992 was used. The fragments obtainedwere separated on agarose gel (1%) in a TAE buffer which made itpossible to reveal a unique band of about 600 bp which was extractedfrom the gel using a DNA purification kit (GeneClean, Bio101 Inc, LaJolla, Calif.). By using a kit leading to the production of blunt endsan the DNA (Amersham, Amersham, Great Britain), the fragments weretreated with the T4 DNA polymerase and ligated at the SmaI site of adigested and dephosphorylated pUC18 plasmid (Norrander et al., 1983,Gene 26: 101-106).

The sequence of 632 bp (vanB probe) corresponding to the insert of therecombinant plasmid (FIG. 1) was determined by the dideoxy chaintermination method (Sanger et al., 1977, Proc Natl. ACad. Sd. USA 74:5463-5467) using T7 DNA polymerase (Pharmacia, Uppsala, Sweden) and{∝-³⁵ S} dATP (Amersham Radiochemical Center, Amersham, Great Britain).

Given that the amplification with the Taq DNA polymerase may lead toerroneous incorporations of nucleotides, the sequence was confirmed asfollows: an oligonucleotide complementary to the positions 513 to 530 ofthe nucleotide sequence shown in FIG. 1 was synthesized by thephosphoramidite method (Organic Chemistry unit, Pasteur Institute,France) and used with the primer V1 to carry out an amplification of avanB fragment by PCR. The PCR product was sequenced directly (Mabilat etal., 1990, Plasmid 23: 27-34) or after the cloning in a pUC18 vector inorder to reveal the identity of the nucleotides with the cloned fragmentobtained with V1 and V2.

A Southern hybridization was carried out according to the method ofJohnson et al., Gene Anal. Technol. 1: 3-8 (1984). The total DNA of thestrains of enterococci (Table 1) was prepared according to the proceduredescribed by Le Bouguenec et al., 1990, J. Bacteriol. 172: 727-734,digested with the enzymes HindIII and KpnI (United States Biochemicalcorporation, Cleveland, Ohio) and resolved on 1% agarose gels. The DNAwas transferred to nylon membranes (Nytran, Schleicher & Schuell,Dassel, Germany) with a transfer apparatus under vacuum (Trans. Vac.TE80, Hoefer Scientific Instruments, San Francisco, Calif.). The probewas obtained by labelling the cloned PCR fragment with a nicktranslation kit (Bethesda Research Laboratories Life Technologies Inc,Gaithersburg, Md.) and {∝-³² p} dCTP (Amersham Radiochemical Center,Amersham, Great Britain). The hybridization was carried out understringent conditions at 68° C. (Johnson et al., 1984, Gene Anal.Technol. 1: 3-8). The membranes were washed at 65° C. in 0.1% SDS-2×SSC

The vanA probe consisted of a PstI fragment of 265 bp internal to thevanA gene (Dukta-Malen S et al., Mol. Gen. Genet. 1990, 224: 364-372).The vanC probe consisted of a EcoRI-HincII fragment of 690 bp internalto the vanC gene (Leclercq R et al., Antimicrob. Agents Chemother. 1992,36: 2005-8. Dukta-Malen S. Et al. Gene, 1992, 112: 53-58). The vanBprobe corresponds to the sequence II.

The amino add sequence deduced for the insert contained in the pUC18plasmid was compared with different protein sequences (FIG. 2): Table 5sunmarises the identity percentages of amino acids when the proteinsequences VanB, VanA, VanC ElDdl, DdlA and DdlB are compared pairwise.Under the conditions of Southern hybridization the cloned fragmenthybridized with the 3.3 kb HindIII-KpnI fragment of E. faecalis V583.The probe does not hybridize with the DNA of a vancomycin-sensitivederivative of V583 or with the DNA of the E. faecalis and E. faeciumstrains sensitive to vancomycin used as reference. The cloned DNAfragment obtained by PCR corresponds to an internal fragment of the geneimplicated in the resistance. This gene codes for the enzyme related tothe D-Ala: D-Ala ligases, called VanB, which might be implicated in thesynthesis of a product substituting for D-Ala-D-Ala.

These tests have made it possible to demonstrate a single group of genesrelated to vanB and responsible for a low- and high-level resistance tovancomycin in the enterococci (Tables 1 and 2).

No hybridization was observed between the VanB probe and the DNA ofstrains sensitive to vancomycin without induction or bearing the vanA orvanC genes or intrinsically resistant.

The complete sequence of the vanB gene was cloned by implementing thefollowing steps:

The plasmid pAT202 was obtained by subcloning in pUC19 a 3.3 kbHindIII-KpnI fragment of the λ recombinant bacteriophage containing thevanB gene. The cloning was performed with restriction endonucleases(Boehringer, Mannheim, Germany and Pharmacia LKB Biotechnology IncUppsala, Sweden), T4 DNA ligase (Boehringer) and alkaline phosphatase(Pharmacia) in conformity with the recommendations of the manufacturer.The nucleotide sequence of the consecutive 1090 bp of pAT202 wasdetermined on both strands by the dideoxy chain termination method(Sanger et al., 1977) using a modified T7 DNA polymerase (AmershamRadiochemical Center, Amersham, Great Britain) and complementaryoligonucleotides of the sequence, synthesized by the methoxyphosphoramidite method (Institute Pasteur, Paris, France). The reactionproducts were resolved by electrophoresis on a 6% denaturingpolyacrylamide gel. The first base pair of the sequence showncorresponds to position 1 (FIG. 1). The potential ribosome binding site(RBS) (Moran et al., Md. Gen. Genet. 186 (1982) 339-346) upstream fromthe ATG initiation codon at position 46 is underlined. The stop codon(TGA) is indicated by an asterisk. The amino acid sequence is alignedwith the first nucleotide of each codon.

The transfer of the vancomycin-resistance character (in 6 isolates ofenterococci out of 17) by conjugation on a filter was observed in E.faecium and E. faecalis strains resistant to low or high concentrationsof antibiotics.

Of the other fragments of about 600 bp amplified from theoligonucleotides V1 and V2, an insert hybridized with the DNA of Vm^(R)or Vm^(S) strains of E. faecalis but not with the DNA of strains of 18other species. This gene codes for a D-Ala: D-ala ligase in E faecalis.Since no other ligase gene was detected in E. faecalis, this gene wascalled ddl.

The cloning and sequencing of the ddl gene inserted in the pUC19 vector(Norrander et al., Gene 26, 1983, 101-106) led to the observation thatthe content of the bases G and C in ddl (37.5%) and the chromosome of E.faecalis (37-39%) were very similar.

Different observations suggest that the vanB gene might have anexogenous origin: (i) The gene may be transferred by conjugation. (ii)The nucleotide sequences related to vanB have not been detected in theDNA of Vm^(S) strains of E. faecalis and E faecium and therepresentatives of 16 other species of Enterococcus (Table III). (iii)The GC base content of the vanB gene differs markedly from that of thechromosome of E. faecalis. (iv) The low level of similarity between Ddlof E. faecalis and VanB (34% identity) indicates that the correspondinggenes have not originated as the result of a recent duplication.

Precursors of Peptidoglycan in E. faecalis Vm^(R) and Vm^(S)

The incubation of E. faecalis V583 before the induction of the Vm^(R) orVm^(S) strains of E faecalis JH2-2 (Jacob and Hobbs, J. Bacteriol. 117,1974, 360-372) with the cell wall inhibitor ramoplanin (9 μg/ml) led tothe accumulation of the cell wall precursorUDP-N-acetyl-muramyl-L-Ala-D-Glu-L-Lys-D-Ala-D-Ala(UDP-Mur-NAc-pentapeptide) which is used in the normal cycle ofpeptidoglycan synthesis.

After the induction of resistance, E. faecalis V583 accumulated threecell wall intermediates when the strain was incubated with ramoplanin(Table IV). These intermediates were identified as beingUDP-MurNAc-pentapeptides, UDP- MurNAc-tetrapeptides lacking theC-terminal D-Ala residue of the UDP-MurNAc-pentapeptide; andpredominantly UDP-MurNAc-tetrapeptide-D-lactate in which the C-terminalD-Ala residue of the UDP-MurNAc-pentapeptide is replaced by D-lactate.The presence of UDP-MurNAc-tetrapeptide-D-lactate suggests that thestrains of the VanA and VanB phenotypes have the same basic resistancemechanism to the glycopeptides; i.e. they synthesize D-lactate which maybe linked to VanB (or VanA) by D-Ala to synthesize D-Ala-D-lactate whichis then incorporated into the peptidoglycan precursor.

The wall precursors were purified by ion exchange chromatography anddesalting by gel filtration. The identification was based on massspectrometry (positive ion electrospray mass spectroscopy), a UVspectrum (for the uracil) automated amino acid analysis after hydrolysisfor 4h and 24h (for muramic acid and the ratios of the amino acids) andthe analysis by specific enzymatic reactions of the terminal residuecarried out by reaction with D,D-carboxypeptidase of Actinomadura R39(Messer and Reynolds, FEMS Micrcbiol. Letter 94, 1992, 195-200).

The total quantity of precursors accumulated in each culture wasapproximately the same. 65 μmol/g of dry weight during an incubationperiod corresponding to 0.6 of the mean synthesis time.

                                      TABLE I                                     __________________________________________________________________________    Bacterial strains                                                                    MIC   Hybridization with                                                      (μg/ml)                                                                          the vanB probe                                                   Strain Vm Te vanA                                                                              vanB                                                                              vanC                                                                              Reference                                            __________________________________________________________________________    E. faecalis                                                                   V583   64 0,5                                                                              -   +   -   Sahm et al (1989)                                    V583-C1                                                                              2  0,5                                                                              -   -   -   D. F. Sahm                                           V583-2 1024                                                                             1,0                                                                              -   +   -   Zarlenga L J (1992)                                  UMH-1  1024                                                                             1,0                                                                              -   +   -   Schwalbe et al (1991)                                ATCC 29212                                                                           2  0,5                                                                              -   -   -                                                        E. faecium                                                                    D366   32 0,5                                                                              -   +   -   Gutmann et al (1992)                                 MT10R  2  0,25                                                                             -   -   -   Gutmann et al (1992)                                 BM4147 1024                                                                             512                                                                              +   -   -   Dutka-Malen et al (1990)                             ATCC 19434                                                                           1  1  -   -   -                                                        E. gallinarum                                                                 BM4174 8  1  -   -   +   Dutka-Malen et al (1992)                             E.casseliflavus                                                                      8  1  -   -   -                                                        __________________________________________________________________________

                                      TABLE II                                    __________________________________________________________________________    Phenotypic and genotypic classes among the Gram-positive                      cocci resistant to vancomycin                                                 PHENOTYPIC                                                                           GENOTYPIC        MIC (μg/ml)                                        CLASS  CLASS (A)                                                                            SPECIES   Vancomycin                                                                          Teicoplanin                                     __________________________________________________________________________    Susceptible                                                                          Susceptible                                                                          Enterococcus spp.(10)                                                                   0.5-2 0.25                                            A      A      E.faecium(6)                                                                            256->1.000                                                                          64->1.000                                       B      B      E.faecium(28)                                                                           4-256 0.5-1                                           B      B      E.faecalis(11)                                                                          4-1024                                                                              0.5-1                                           C      C      E.gallinarum(3)                                                                         8     1                                               C      NC     E.casseliflavus(2)                                                                      4-8   0.5-1                                           NC     NC     Lactobacillus spp.(8)                                                                   >1.000                                                                              >1.000                                          NC     NC     Leuconostoc. sp.(1)                                                                     >1.000                                                                              >1.000                                          NC     NC     Pediococcus spp.(2)                                                                     >1.000                                                                              >1.000                                          NC     NC     E.rhusiopathiae(1)                                                                      >1.000                                                                              >1.000                                          __________________________________________________________________________     .sup.(a) A: hybridization with the vanA probe; B: hybridization with the      vanB probe                                                                    C: hybridization with the vanC probe; NC: not classed                    

                  TABLE III                                                       ______________________________________                                        Results of hybridization experiments                                                              Number                                                               Resistance                                                                             of        Hybridization with probe                        Species    phenotype                                                                              strains tested                                                                          vanB ddl (En. faecalis)                         ______________________________________                                        En. faecalis                                                                             Vm.sup.R, Te.sup.S                                                                     11        +    +                                                     Vm.sup.S, Te.sup.S                                                                     5         -    +                                          En. faecium                                                                              Vm.sup.R, Te.sup.R                                                                     6         -    -                                                     Vm.sup.R, Te.sup.S                                                                     28        +    -                                                     Vm.sup.S, Te.sup.S                                                                     4         -    -                                          En. gallinarum                                                                           Vm.sup.R, Te.sup.S                                                                     3         -    -                                          En. casseliflavus                                                                        Vm.sup.r, Te.sup.S                                                                     2         -    -                                          En. spp.   Vm.sup.S, Te.sup.S                                                                     15        -    -                                          (15 species.sup.a)                                                            ______________________________________                                         .sup.a Types of strains En. avium, En. cecorum, En. columbae, En. dispar,     En. durans, En. flavescens, En. hirae, En. malodorarus, En. mundrii, En.      pseudoavium, En. raffinosus, En. saccharolyticus, En. seriolicida, En.        solitarius et En. sulfureus.                                             

                                      TABLE IV                                    __________________________________________________________________________    Peptidoglycan precursors in Vm.sup.S or Vm.sup.R strains of En faecalis                        Quantity of precursor (%) in En faecalis                     Peptidoglycan precursor                                                                        JH2-2                                                                             V583 not induced                                                                      V583 induced                                     __________________________________________________________________________    UDP--MurNAc--L--Ala--D--Glu--                                                                  100 100     14                                               L--Lys--D--Ala--D--Ala                                                        UDP--MurNAc--L--Ala--D--Glu--                                                                  0   0        7                                               L--Lys--D--Ala                                                                UDP--MurNAc--L--Ala--D--Glu--                                                                  0   0       79                                               L--Lys--D--Ala--D--lactate                                                    __________________________________________________________________________

                  TABLE V                                                         ______________________________________                                        Sequence identity between the amino acid sequnces of VanB,                    Ddl of En. faecalis V583 and D--Ala: D--Ala ligases.sup.a                     Compared Percentage identity with respect to:                                 sequence.sup.b                                                                         VanB    VanC    EfDdl EcDdlA                                                                              StDdlA                                                                              EcDdlB                             ______________________________________                                        VanA     76      38      32    38    37    30                                 VanB             38      34    38    38    32                                 VanC                     34    34    34    36                                 EfDdl                          40    40    34                                 EcDdlA                               90    35                                 StDdlA                                     36                                 ______________________________________                                         .sup.a Identity of pairs of sequences derived from the alignment of FIG.      .sup.b Ec, E.coli; Ef, En.faecalis; St, S.typhimurium                    

    __________________________________________________________________________    SEQUENCE LISTING                                                              (1) GENERAL INFORMATION:                                                      (iii) NUMBER OF SEQUENCES: 17                                                 (2) INFORMATION FOR SEQ ID NO:1:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 1140 base pairs                                                   (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: double                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA (genomic)                                             (ix) FEATURE:                                                                 (A) NAME/KEY: CDS                                                             (B) LOCATION: 68..1093                                                        (xi) SEQUENCE DESCRIPTION: SEQ ID NO:1:                                       GAGCGTGTGCTGCGAGATACCACAGAAAACAATCAGAATTGTCTTAACTTTGAAAGGAGT60                TTACAGCATGAATAAAATAAAAGTCGCAATTATCTTCGGCGGTTGCTCG109                          MetAsnLysIleLysValAlaIleIlePheGlyGlyCysSer                                    1510                                                                          GAGGAACATGATGTGTCGGTAAAATCCGCAATAGAAATTGCTGCGAAC157                           GluGluHisAspValSerValLysSerAlaIleGluIleAlaAlaAsn                              15202530                                                                      ATTAATACTGAAAAATTCGATCCGCACTACATCGGAATTACAAAAAAC205                           IleAsnThrGluLysPheAspProHisTyrIleGlyIleThrLysAsn                              354045                                                                        GGCGTATGGAAGCTATGCAAGAAGCCATGTACGGAATGGGAAGCCGAT253                           GlyValTrpLysLeuCysLysLysProCysThrGluTrpGluAlaAsp                              505560                                                                        AGTCTCCCCGCCATATTCTCCCCGGATAGGAAAACGCATGGTCTGCTT301                           SerLeuProAlaIlePheSerProAspArgLysThrHisGlyLeuLeu                              657075                                                                        GTCATGAAAGAAAGAGAATACGAAACTCGGCGTATTGACGTGGCTTTC349                           ValMetLysGluArgGluTyrGluThrArgArgIleAspValAlaPhe                              808590                                                                        CCGGTTTTGCATGGCAAATGCGGGGAGGATGGTGCGATACAGGGTCTG397                           ProValLeuHisGlyLysCysGlyGluAspGlyAlaIleGlnGlyLeu                              95100105110                                                                   TTTGAATTGTCTGGTATCCCCTATGTAGGCTGCGATATTCAAAGCTCC445                           PheGluLeuSerGlyIleProTyrValGlyCysAspIleGlnSerSer                              115120125                                                                     GCAGCTTGCATGGACAAATCACTGGCCTACATTCTTACAAAAAATGCG493                           AlaAlaCysMetAspLysSerLeuAlaTyrIleLeuThrLysAsnAla                              130135140                                                                     GGCATCGCCGTCCCCGAATTTCAAATGATTGAAAAAGGTGACAAACCG541                           GlyIleAlaValProGluPheGlnMetIleGluLysGlyAspLysPro                              145150155                                                                     GAGGCGAGGACGCTTACCTACCCTGTCTTTGTGAAGCCGGCACGGTCA589                           GluAlaArgThrLeuThrTyrProValPheValLysProAlaArgSer                              160165170                                                                     GGTTCGTCCTTTGGCGTAACCAAAGTAAACAGTACGGAAGAACTAAAC637                           GlySerSerPheGlyValThrLysValAsnSerThrGluGluLeuAsn                              175180185190                                                                  GCTGCGATAGAAGCAGCAGGACAATATGATGGAAAAATCTTAATTGAG685                           AlaAlaIleGluAlaAlaGlyGlnTyrAspGlyLysIleLeuIleGlu                              195200205                                                                     CAAGCGATTTCGGGCTGTGAGGTCGGCTGCGCGGTCATGGGAAACGAG733                           GlnAlaIleSerGlyCysGluValGlyCysAlaValMetGlyAsnGlu                              210215220                                                                     GATGATTTGATTGTCGGCGAAGTGGATCAAATCCGGTTGAGCCACGGT781                           AspAspLeuIleValGlyGluValAspGlnIleArgLeuSerHisGly                              225230235                                                                     ATCTTCCGCATCCATCAGGAAAACGAGCCGGAAAAAGGCTCAGAGAAT829                           IlePheArgIleHisGlnGluAsnGluProGluLysGlySerGluAsn                              240245250                                                                     GCGATGATTATCGTTCCAGCAGACATTCCGGTCGAGGAACGAAATCGG877                           AlaMetIleIleValProAlaAspIleProValGluGluArgAsnArg                              255260265270                                                                  GTGCAAGAAACGGCAAAGAAAGTATATCGGGTGCTTGGATGCAGAGGG925                           ValGlnGluThrAlaLysLysValTyrArgValLeuGlyCysArgGly                              275280285                                                                     CTTGCTCGTGTTGATCTTTTTTTGCAGGAGGATGGCGGCATCGTTCTA973                           LeuAlaArgValAspLeuPheLeuGlnGluAspGlyGlyIleValLeu                              290295300                                                                     AACGAGGTCAATACCCTGCCCGGTTTTACATCGTACAGCCGCTATCCA1021                          AsnGluValAsnThrLeuProGlyPheThrSerTyrSerArgTyrPro                              305310315                                                                     CGCATGGCGGCTGCCGCAGGAATCACGCTTCCCGCACTAATTGACAGC1069                          ArgMetAlaAlaAlaAlaGlyIleThrLeuProAlaLeuIleAspSer                              320325330                                                                     CTGATTACATTGGCGATAGAGAGGTGACCCGTATGGAAAATGGTTTTTTGTTTT1123                    LeuIleThrLeuAlaIleGluArg                                                      335340                                                                        TTAGATGAAATGTTGCA1140                                                         (2) INFORMATION FOR SEQ ID NO:2:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 342 amino acids                                                   (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: protein                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:2:                                       MetAsnLysIleLysValAlaIleIlePheGlyGlyCysSerGluGlu                              151015                                                                        HisAspValSerValLysSerAlaIleGluIleAlaAlaAsnIleAsn                              202530                                                                        ThrGluLysPheAspProHisTyrIleGlyIleThrLysAsnGlyVal                              354045                                                                        TrpLysLeuCysLysLysProCysThrGluTrpGluAlaAspSerLeu                              505560                                                                        ProAlaIlePheSerProAspArgLysThrHisGlyLeuLeuValMet                              65707580                                                                      LysGluArgGluTyrGluThrArgArgIleAspValAlaPheProVal                              859095                                                                        LeuHisGlyLysCysGlyGluAspGlyAlaIleGlnGlyLeuPheGlu                              100105110                                                                     LeuSerGlyIleProTyrValGlyCysAspIleGlnSerSerAlaAla                              115120125                                                                     CysMetAspLysSerLeuAlaTyrIleLeuThrLysAsnAlaGlyIle                              130135140                                                                     AlaValProGluPheGlnMetIleGluLysGlyAspLysProGluAla                              145150155160                                                                  ArgThrLeuThrTyrProValPheValLysProAlaArgSerGlySer                              165170175                                                                     SerPheGlyValThrLysValAsnSerThrGluGluLeuAsnAlaAla                              180185190                                                                     IleGluAlaAlaGlyGlnTyrAspGlyLysIleLeuIleGluGlnAla                              195200205                                                                     IleSerGlyCysGluValGlyCysAlaValMetGlyAsnGluAspAsp                              210215220                                                                     LeuIleValGlyGluValAspGlnIleArgLeuSerHisGlyIlePhe                              225230235240                                                                  ArgIleHisGlnGluAsnGluProGluLysGlySerGluAsnAlaMet                              245250255                                                                     IleIleValProAlaAspIleProValGluGluArgAsnArgValGln                              260265270                                                                     GluThrAlaLysLysValTyrArgValLeuGlyCysArgGlyLeuAla                              275280285                                                                     ArgValAspLeuPheLeuGlnGluAspGlyGlyIleValLeuAsnGlu                              290295300                                                                     ValAsnThrLeuProGlyPheThrSerTyrSerArgTyrProArgMet                              305310315320                                                                  AlaAlaAlaAlaGlyIleThrLeuProAlaLeuIleAspSerLeuIle                              325330335                                                                     ThrLeuAlaIleGluArg                                                            340                                                                           (2) INFORMATION FOR SEQ ID NO:3:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 589 base pairs                                                    (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: other nucleic acid                                        (xi) SEQUENCE DESCRIPTION: SEQ ID NO:3:                                       TCTGTTTGAATTGTCTGGTATCCCCTATGTAGGCTGCGATATTCAAAGCTCCGCAGCTTG60                CATGGACAAATCACTGGCCTACATTCTTACAAAAAATGCGGGCATCGCCGTCCCCGAATT120               TCAAATGATTGAAAAAGGTGACAAACCGGAGGCGAGGACGCTTACCTACCCTGTCTTTGT180               GAAGCCGGCACGGTCAGGTTCGTCCTTTGGCGTAACCAAAGTAAACAGTACGGAAGAACT240               AAACGCTGCGATAGAAGCAGCAGGACAATATGATGGAAAAATCTTAATTGAGCAAGCGAT300               TTCGGGCTGTGAGGTCGGCTGCGCGGTCATGGGAAACGAGGATGATTTGATTGTCGGCGA360               AGTGGATCAAATCCGGTTGAGCCACGGTATCTTCCGCATCCATCAGGAAAACGAGCCGGA420               AAAAGGCTCAGAGAATGCGATGATTATCGTTCCAGCAGACATTCCGGTCGAGGAACGAAA480               TCGGGTGCAAGAAACGGCAAAGAAAGTATATCGGGTGCTTGGATGCAGAGGGCTTGCTCG540               TGTTGATCTTTTTTTGCAGGAGGATGGCGGCATCGTTCTAAACGAGGTC589                          (2) INFORMATION FOR SEQ ID NO:4:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 17 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: other nucleic acid                                        (xi) SEQUENCE DESCRIPTION: SEQ ID NO:4:                                       ATGGGAAGCCGATAGTC17                                                           (2) INFORMATION FOR SEQ ID NO:5:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 17 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: other nucleic acid                                        (xi) SEQUENCE DESCRIPTION: SEQ ID NO:5:                                       GATTTCGTTCCTCGACC17                                                           (2) INFORMATION FOR SEQ ID NO:6:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 343 amino acids                                                   (B) TYPE: amino acid                                                          (C) STRANDEDNESS: Not Relevant                                                (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: protein                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:6:                                       MetAsnArgIleLysValAlaIleLeuPheGlyGlyCysSerGluGlu                              151015                                                                        HisAspValSerValLysSerAlaIleGluIleAlaAlaAsnIleAsn                              202530                                                                        LysGluLysTyrGluProLeuTyrIleGlyIleThrLysSerGlyVal                              354045                                                                        TrpLysMetCysGluLysProCysAlaGluTrpGluAsnAspAsnCys                              505560                                                                        TyrSerAlaValLeuSerProAspLysLysMetHisGlyLeuLeuVal                              65707580                                                                      LysLysAsnHisGluTyrGluIleAsnHisValAspValAlaPheSer                              859095                                                                        AlaLeuHisGlyLysSerGlyGluAspGlySerIleGlnGlyLeuPhe                              100105110                                                                     GluLeuSerGlyIleProPheValGlyCysAspIleGlnSerSerAla                              115120125                                                                     IleCysMetAspLysSerLeuThrTyrIleValAlaLysAsnAlaGly                              130135140                                                                     IleAlaThrProAlaPheTrpValIleAsnLysAspAspArgProVal                              145150155160                                                                  AlaAlaThrPheThrTyrProValPheValLysProAlaArgSerGly                              165170175                                                                     SerSerPheGlyValLysLysValAsnSerAlaAspGluLeuAspTyr                              180185190                                                                     AlaIleGluSerAlaArgGlnTyrAspSerLysIleLeuIleGluGln                              195200205                                                                     AlaValSerGlyCysGluValGlyCysAlaValLeuGlyAsnSerAla                              210215220                                                                     AlaLeuValValGlyGluValAspGlnIleArgLeuGlnTyrGlyIle                              225230235240                                                                  PheArgIleHisGlnGluValGluProGluLysGlySerGluAsnAla                              245250255                                                                     ValIleThrValProAlaAspLeuSerAlaGluGluArgGlyArgIle                              260265270                                                                     GlnGluThrAlaLysLysIleTyrLysAlaLeuGlyCysArgGlyLeu                              275280285                                                                     AlaArgValAspMetPheLeuGlnAspAsnGlyArgIleValLeuAsn                              290295300                                                                     GluValAsnThrLeuProGlyPheThrSerTyrSerArgTyrProArg                              305310315320                                                                  MetMetAlaAlaAlaGlyIleAlaLeuProGluLeuIleAspArgLeu                              325330335                                                                     IleValLeuAlaLeuLysGly                                                         340                                                                           (2) INFORMATION FOR SEQ ID NO:7:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 343 amino acids                                                   (B) TYPE: amino acid                                                          (C) STRANDEDNESS: Not Relevant                                                (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: protein                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:7:                                       MetLysLysIleAlaValLeuPheGlyGlyAsnSerProGluTyrSer                              151015                                                                        ValSerLeuThrSerAlaAlaSerValIleGlnAlaIleAspProLeu                              202530                                                                        LysTyrGluValMetThrIleGlyIleAlaProThrMetAspTrpTyr                              354045                                                                        TrpTyrGlnGlyAsnLeuAlaAsnValArgAsnAspThrTrpLeuGlu                              505560                                                                        AspHisLysAsnCysHisGlnLeuThrPheSerSerGlnGlyPheIle                              65707580                                                                      LeuGlyGluLysArgIleValProAspValLeuPheProValLeuHis                              859095                                                                        GlyLysTyrGlyGluAspGlyCysIleGlnGlyLeuLeuGluLeuMet                              100105110                                                                     AsnLeuProTyrValGlyCysHisValAlaAlaSerAlaLeuCysMet                              115120125                                                                     AsnLysTrpLeuLeuHisGlnLeuAlaAspThrMetGlyIleAlaSer                              130135140                                                                     AlaProThrLeuLeuLeuSerArgTyrGluAsnAspProAlaThrIle                              145150155160                                                                  AspArgPheIleGlnAspHisGlyPheProIlePheIleLysProAsn                              165170175                                                                     GluAlaGlySerSerLysGlyIleThrLysValThrAspLysThrAla                              180185190                                                                     LeuGlnSerAlaLeuThrThrAlaPheAlaTyrGlySerThrValLeu                              195200205                                                                     IleGlnLysAlaIleAlaGlyIleGluIleGlyCysGlyIleLeuGly                              210215220                                                                     AsnGluGlnLeuThrIleGlyAlaCysAspAlaIleSerLeuValAsp                              225230235240                                                                  GlyPhePheAspPheGluGluLysTyrGlnLeuIleSerAlaThrIle                              245250255                                                                     ThrValProAlaProLeuProLeuAlaLeuGluSerGlnIleLysGlu                              260265270                                                                     GlnAlaGlnLeuLeuTyrArgAsnLeuGlyLeuThrGlyLeuAlaArg                              275280285                                                                     IleAspPhePheValThrAsnGlnGlyAlaIleTyrLeuAsnGluIle                              290295300                                                                     AsnThrMetProGlyPheThrGlyHisSerArgTyrProAlaMetMet                              305310315320                                                                  AlaGluValGlyLeuSerTyrGluIleLeuValGluGlnLeuIleAla                              325330335                                                                     LeuAlaGluGluAspLysArg                                                         340                                                                           (2) INFORMATION FOR SEQ ID NO:8:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 348 amino acids                                                   (B) TYPE: amino acid                                                          (C) STRANDEDNESS: Not Relevant                                                (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: protein                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:8:                                       LeuLysIleIleLeuLeuTyrGlyGlyArgSerGluGluHisAspVal                              151015                                                                        SerValLeuSerAlaTyrSerValLeuAsnAlaIleTyrTyrLysTyr                              202530                                                                        TyrGlnValGlnLeuValPheIleSerLysAspGlyGlnTrpValLys                              354045                                                                        GlyProLeuLeuSerGluArgProGlnAsnLysGluValLeuHisLeu                              505560                                                                        ThrTrpAlaGlnThrProGluGluThrGlyGluPheSerGlyLysArg                              65707580                                                                      IleSerProSerGluIleTyrGluGluGluAlaIleValPheProVal                              859095                                                                        LeuHisGlyProAsnGlyGluAspGlySerIleGlnGlyPheMetGlu                              100105110                                                                     ThrIleAsnMetProTyrValGlyAlaGlyValLeuAlaSerAlaAsn                              115120125                                                                     AlaMetAspLysIleMetThrLysValLeuLeuGlnThrValGlyIle                              130135140                                                                     ProGlnValProPheValProValLeuArgSerAspTrpLysGlyAsn                              145150155160                                                                  ProLysGluValThrGluLysCysGluGlySerLeuIleTyrProVal                              165170175                                                                     PheValLysProAlaAsnMetGlySerSerValGlyIleSerLysVal                              180185190                                                                     GluAsnArgAspGluLeuGlnGluAlaLeuGluGluAlaPheArgTyr                              195200205                                                                     AspAlaArgAlaIleValGluGlnGlyIleGluAlaArgGluIleGlu                              210215220                                                                     ValAlaIleLeuGlyAsnGluAspValArgThrThrLeuProGlyGlu                              225230235240                                                                  ValValLysAspValAlaPheTyrAspTyrAspAlaLysTyrIleAsn                              245250255                                                                     AsnThrIleGluMetGlnIleProAlaHisValProGluGluValAla                              260265270                                                                     HisGlnAlaGlnGluTyrAlaLysLysAlaTyrIleMetLeuAspGly                              275280285                                                                     SerGlyLeuSerArgCysAspPhePheLeuThrSerLysAsnGluLeu                              290295300                                                                     PheLeuAsnGluLeuAsnThrMetProGlyPheThrProPheSerMet                              305310315320                                                                  TyrProLeuLeuTrpGluAsnMetGlyLeuLysTyrSerAspLeuIle                              325330335                                                                     GluGluLeuIleGlnLeuAlaLeuAsnArgPheLys                                          340345                                                                        (2) INFORMATION FOR SEQ ID NO:9:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 364 amino acids                                                   (B) TYPE: amino acid                                                          (C) STRANDEDNESS: Not Relevant                                                (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: protein                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:9:                                       MetGluLysLeuArgValGlyIleValPheGlyGlyLysSerAlaGlu                              151015                                                                        HisGluValSerLeuGlnSerAlaLysAsnIleValAspAlaIleAsp                              202530                                                                        LysSerArgPheAspValValLeuLeuGlyIleAspLysGlnGlyGln                              354045                                                                        TrpHisValSerAspAlaSerAsnTyrLeuLeuAsnAlaAspAspPro                              505560                                                                        AlaHisIleAlaLeuArgProSerAlaThrSerLeuAlaGlnValPro                              65707580                                                                      GlyLysHisGluHisGlnLeuIleAspAlaGlnAsnGlyGlnProLeu                              859095                                                                        ProThrValAspValIlePheProIleValHisGlyThrLeuGlyGlu                              100105110                                                                     AspGlySerLeuGlnGlyMetLeuArgValAlaAsnLeuProPheVal                              115120125                                                                     GlySerAspValLeuAlaSerAlaAlaCysMetAspLysAspValThr                              130135140                                                                     LysArgLeuLeuArgAspAlaGlyLeuAsnIleAlaProPheIleThr                              145150155160                                                                  LeuThrArgAlaAsnArgHisAsnIleSerPheAlaGluValGluSer                              165170175                                                                     LysLeuGlyLeuProLeuPheValLysProAlaAsnGlnGlySerSer                              180185190                                                                     ValGlyValSerLysValThrSerGluGluGlnTyrAlaIleAlaVal                              195200205                                                                     AspLeuAlaPheGluPheAspHisLysValIleValGluGlnGlyIle                              210215220                                                                     LysGlyArgGluIleGluCysAlaValLeuGlyAsnAspAsnProGln                              225230235240                                                                  AlaSerThrCysGlyGluIleValLeuThrSerAspPheTyrAlaTyr                              245250255                                                                     AspThrLysTyrIleAspGluAspGlyAlaLysValValValProAla                              260265270                                                                     AlaIleAlaProGluIleAsnAspLysIleArgAlaIleAlaValGln                              275280285                                                                     AlaTyrGlnThrLeuGlyCysAlaGlyMetAlaArgValAspValPhe                              290295300                                                                     LeuThrProGluAsnGluValValIleAsnGluIleAsnThrLeuPro                              305310315320                                                                  GlyPheThrAsnIleSerMetTyrProLysLeuTrpGlnAlaSerGly                              325330335                                                                     LeuGlyTyrThrAspLeuIleThrArgLeuIleGluLeuAlaLeuGlu                              340345350                                                                     ArgHisAlaAlaAsnAsnAlaLeuLysThrThrMet                                          355360                                                                        (2) INFORMATION FOR SEQ ID NO:10:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 364 amino acids                                                   (B) TYPE: amino acid                                                          (C) STRANDEDNESS: Not Relevant                                                (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: protein                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:10:                                      MetAlaLysLeuArgValGlyIleValPheGlyGlyLysSerAlaGlu                              151015                                                                        HisGluValSerLeuGlnSerAlaLysAsnIleValAspAlaIleAsp                              202530                                                                        LysThrArgPheAspValValLeuLeuGlyIleAspLysAlaGlyGln                              354045                                                                        TrpHisValAsnAspAlaGluAsnTyrLeuGlnAsnAlaAspAspPro                              505560                                                                        AlaHisIleAlaLeuArgProSerAlaIleSerLeuAlaGlnValPro                              65707580                                                                      GlyLysHisGlnHisGlnLeuIleAsnAlaGlnAsnGlyGlnProLeu                              859095                                                                        ProThrValAspValIlePheProIleValHisGlyThrLeuGlyGlu                              100105110                                                                     AspGlySerLeuGlnGlyMetLeuArgValAlaAsnLeuProPheVal                              115120125                                                                     GlySerAspValLeuSerSerAlaAlaCysMetAspLysAspValAla                              130135140                                                                     LysArgLeuLeuArgAspAlaGlyLeuAsnIleAlaProPheIleThr                              145150155160                                                                  LeuThrArgThrAsnArgHisAlaPheSerPheAlaGluValGluSer                              165170175                                                                     ArgLeuGlyLeuProLeuPheValLysProAlaAsnGlnGlySerSer                              180185190                                                                     ValGlyValSerLysValAlaAsnGluAlaGlnTyrGlnGlnAlaVal                              195200205                                                                     AlaLeuAlaPheGluPheAspHisLysValValValGluGlnGlyIle                              210215220                                                                     LysGlyArgGluIleGluCysAlaValLeuGlyAsnAspAsnProGln                              225230235240                                                                  AlaSerThrCysGlyGluIleValLeuAsnSerGluPheTyrAlaTyr                              245250255                                                                     AspThrLysTyrIleAspAspAsnGlyAlaGlnValValValProAla                              260265270                                                                     GlnIleProSerGluValAsnAspLysIleArgAlaIleAlaIleGln                              275280285                                                                     AlaTyrGlnThrLeuGlyCysAlaGlyMetAlaArgValAspValPhe                              290295300                                                                     LeuThrAlaAspAsnGluValValIleAsnGluIleAsnThrLeuPro                              305310315320                                                                  GlyPheThrAsnIleSerMetTyrProLysLeuTrpGlnAlaSerGly                              325330335                                                                     LeuGlyTyrThrAspLeuIleSerArgLeuIleGluLeuAlaLeuGlu                              340345350                                                                     ArgHisThrAlaAsnAsnAlaLeuLysThrThrMet                                          355360                                                                        (2) INFORMATION FOR SEQ ID NO:11:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 306 amino acids                                                   (B) TYPE: amino acid                                                          (C) STRANDEDNESS: Not Relevant                                                (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: protein                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:11:                                      MetThrAspLysIleAlaValLeuLeuGlyGlyThrSerAlaGluArg                              151015                                                                        GluValSerLeuAsnSerGlyAlaAlaValLeuAlaGlyLeuArgGlu                              202530                                                                        GlyGlyIleAspAlaTyrProValAspProLysGluValAspValThr                              354045                                                                        GlnLeuLysSerMetGlyPheGlnLysValPheIleAlaLeuHisGly                              505560                                                                        ArgGlyGlyGluAspGlyThrLeuGlnGlyMetLeuGluLeuMetGly                              65707580                                                                      LeuProTyrThrGlySerGlyValMetAlaSerAlaLeuSerMetAsp                              859095                                                                        LysLeuArgSerLysLeuLeuTrpGlnGlyAlaGlyLeuProValAla                              100105110                                                                     ProTrpValAlaLeuThrArgAlaGluPheGluLysGlyLeuSerAsp                              115120125                                                                     LysGlnLeuAlaGluIleSerAlaLeuGlyLeuProValIleValLys                              130135140                                                                     ProSerArgGluGlySerSerValGlyMetSerLysValValAlaGlu                              145150155160                                                                  AsnAlaLeuGlnAspAlaLeuArgLeuAlaPheGlnHisAspGluGlu                              165170175                                                                     ValLeuIleGluLysTrpLeuSerGlyProGluPheThrValAlaIle                              180185190                                                                     LeuGlyGluGluIleLeuProSerIleArgIleGlnProSerGlyThr                              195200205                                                                     PheTyrAspTyrGluAlaLysTyrLeuSerAspGluThrGlnTyrPhe                              210215220                                                                     CysProAlaGlyLeuGluAlaSerGlnGluAlaAsnLeuGlnAlaLeu                              225230235240                                                                  ValLeuLysAlaTrpThrThrLeuGlyCysLysGlyTrpGlyArgIle                              245250255                                                                     AspValMetLeuAspSerAspGlyGlnPheTyrLeuLeuGluAlaAsn                              260265270                                                                     ThrSerProGlyMetThrSerHisSerLeuValProMetAlaAlaArg                              275280285                                                                     GlnAlaGlyMetSerPheSerGlnLeuValValArgIleLeuGluLeu                              290295300                                                                     AlaAsp                                                                        305                                                                           (2) INFORMATION FOR SEQ ID NO:12:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 24 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: other nucleic acid                                        (xi) SEQUENCE DESCRIPTION: SEQ ID NO:12:                                      GGNGARGAYGGNWSNYTNCARGGN24                                                    (2) INFORMATION FOR SEQ ID NO:13:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 21 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: other nucleic acid                                        (xi) SEQUENCE DESCRIPTION: SEQ ID NO:13:                                      AAYACNHTNCCNGGNTTTACN21                                                       (2) INFORMATION FOR SEQ ID NO:14:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 23 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: other nucleic acid                                        (xi) SEQUENCE DESCRIPTION: SEQ ID NO:14:                                      GGNGARGAYGGNRSNHTNCARGG23                                                     (2) INFORMATION FOR SEQ ID NO:15:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 20 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: other nucleic acid                                        (xi) SEQUENCE DESCRIPTION: SEQ ID NO:15:                                      TGRAANCCNGGNADNGTRTT20                                                        (2) INFORMATION FOR SEQ ID NO:16:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 1079 base pairs                                                   (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: double                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA (genomic)                                             (ix) FEATURE:                                                                 (A) NAME/KEY: CDS                                                             (B) LOCATION: 33..1076                                                        (xi) SEQUENCE DESCRIPTION: SEQ ID NO:16:                                      AAAGACAGGAAAGAAACTAGGAGGACAAGCATTTGAAGATTATTTTGTTGTAT53                       LeuLysIleIleLeuLeuTyr                                                         345                                                                           GGCGGCAGAAGTGAAGAGCACGATGTGTCTGTTTTGTCTGCATATTCC101                           GlyGlyArgSerGluGluHisAspValSerValLeuSerAlaTyrSer                              350355360365                                                                  GTTTTAAATGCAATCTATTATAAATATTATCAAGTACAGTTAGTCTTT149                           ValLeuAsnAlaIleTyrTyrLysTyrTyrGlnValGlnLeuValPhe                              370375380                                                                     ATTAGTAAAGACGGTCAATGGGTAAAAGGCCCTCTTTTATCTGAACGA197                           IleSerLysAspGlyGlnTrpValLysGlyProLeuLeuSerGluArg                              385390395                                                                     CCACAAAATAAAGAAGTTTTACATTTAACTTGGGCACAAACACCTGAA245                           ProGlnAsnLysGluValLeuHisLeuThrTrpAlaGlnThrProGlu                              400405410                                                                     GAAACAGGCGAATTTTCAGGAAAACGAATCAGTCCTTCGGAAATTTAT293                           GluThrGlyGluPheSerGlyLysArgIleSerProSerGluIleTyr                              415420425                                                                     GAAGAAGAAGCGATTGTTTTCCCTGTTTTACATGGGCCAAATGGTGAA341                           GluGluGluAlaIleValPheProValLeuHisGlyProAsnGlyGlu                              430435440445                                                                  GATGGAACAATTCAAGGATTCATGGAAACCATTAATATGCCTTATGTA389                           AspGlyThrIleGlnGlyPheMetGluThrIleAsnMetProTyrVal                              450455460                                                                     GGCGCGGGTGTCTTAGCTAGCGTTAACGCAATGGACAAAATCATGACG437                           GlyAlaGlyValLeuAlaSerValAsnAlaMetAspLysIleMetThr                              465470475                                                                     AAATATCTTTTACAAACTGTTGGCATTCCACAAGTACCATTCGTGCCA485                           LysTyrLeuLeuGlnThrValGlyIleProGlnValProPheValPro                              480485490                                                                     GTTTTAAGAAGTGACTGGAAAGGAAATCCAAAAGAAGTCTTTGAAAAA533                           ValLeuArgSerAspTrpLysGlyAsnProLysGluValPheGluLys                              495500505                                                                     TGTGAAGGTTCTTTAATTTATCCGGTCTTTGTTAAACCTGCCAATATG581                           CysGluGlySerLeuIleTyrProValPheValLysProAlaAsnMet                              510515520525                                                                  GGTTCTAGTGTCGGAATTAGCAAAGTGGAAAATCGTGAAGAATTGCAA629                           GlySerSerValGlyIleSerLysValGluAsnArgGluGluLeuGln                              530535540                                                                     GAAGCATTGGAAGAAGCTTTCCGTTATGATGCCCGAGCAATTGTTGAA677                           GluAlaLeuGluGluAlaPheArgTyrAspAlaArgAlaIleValGlu                              545550555                                                                     CAAGGGATCGAAGCACGTGAAATTGAAGTAGCCATTTTAGGAAATGAA725                           GlnGlyIleGluAlaArgGluIleGluValAlaIleLeuGlyAsnGlu                              560565570                                                                     GATGTCCGTACGACTTTACCTGGTGAAGTGGTGAAAGATGTCGCTTTC773                           AspValArgThrThrLeuProGlyGluValValLysAspValAlaPhe                              575580585                                                                     TATGATTATGATGCAAAATACATCAATAACACGATTGAAATGCAAATC821                           TyrAspTyrAspAlaLysTyrIleAsnAsnThrIleGluMetGlnIle                              590595600605                                                                  CCAGCGCATGTTCCAGAAGAAGTAGCTCATCAAGCGCAAGAATACGCT869                           ProAlaHisValProGluGluValAlaHisGlnAlaGlnGluTyrAla                              610615620                                                                     AAAAAAGCGTATATTATGTTAGATGGAAGTGGCTTAAGTCGCTGTGAT917                           LysLysAlaTyrIleMetLeuAspGlySerGlyLeuSerArgCysAsp                              625630635                                                                     TTCTTCTTAACAAGCAAAAACGAATTATTCCTGAATGAATTGAACACC965                           PhePheLeuThrSerLysAsnGluLeuPheLeuAsnGluLeuAsnThr                              640645650                                                                     ATGCCTGGTTTTACTGACTTTAGTATGTATCCTTTACTGTGGGAAAAT1013                          MetProGlyPheThrAspPheSerMetTyrProLeuLeuTrpGluAsn                              655660665                                                                     ATGGGCTTGAAATACAGTGATTTAATTGAGGAACTGATTCAGTTAGCT1061                          MetGlyLeuLysTyrSerAspLeuIleGluGluLeuIleGlnLeuAla                              670675680685                                                                  TTGAATCGTTTTAAATAA1079                                                        LeuAsnArgPheLys                                                               690                                                                           (2) INFORMATION FOR SEQ ID NO:17:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 348 amino acids                                                   (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: protein                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:17:                                      LeuLysIleIleLeuLeuTyrGlyGlyArgSerGluGluHisAspVal                              151015                                                                        SerValLeuSerAlaTyrSerValLeuAsnAlaIleTyrTyrLysTyr                              202530                                                                        TyrGlnValGlnLeuValPheIleSerLysAspGlyGlnTrpValLys                              354045                                                                        GlyProLeuLeuSerGluArgProGlnAsnLysGluValLeuHisLeu                              505560                                                                        ThrTrpAlaGlnThrProGluGluThrGlyGluPheSerGlyLysArg                              65707580                                                                      IleSerProSerGluIleTyrGluGluGluAlaIleValPheProVal                              859095                                                                        LeuHisGlyProAsnGlyGluAspGlyThrIleGlnGlyPheMetGlu                              100105110                                                                     ThrIleAsnMetProTyrValGlyAlaGlyValLeuAlaSerValAsn                              115120125                                                                     AlaMetAspLysIleMetThrLysTyrLeuLeuGlnThrValGlyIle                              130135140                                                                     ProGlnValProPheValProValLeuArgSerAspTrpLysGlyAsn                              145150155160                                                                  ProLysGluValPheGluLysCysGluGlySerLeuIleTyrProVal                              165170175                                                                     PheValLysProAlaAsnMetGlySerSerValGlyIleSerLysVal                              180185190                                                                     GluAsnArgGluGluLeuGlnGluAlaLeuGluGluAlaPheArgTyr                              195200205                                                                     AspAlaArgAlaIleValGluGlnGlyIleGluAlaArgGluIleGlu                              210215220                                                                     ValAlaIleLeuGlyAsnGluAspValArgThrThrLeuProGlyGlu                              225230235240                                                                  ValValLysAspValAlaPheTyrAspTyrAspAlaLysTyrIleAsn                              245250255                                                                     AsnThrIleGluMetGlnIleProAlaHisValProGluGluValAla                              260265270                                                                     HisGlnAlaGlnGluTyrAlaLysLysAlaTyrIleMetLeuAspGly                              275280285                                                                     SerGlyLeuSerArgCysAspPhePheLeuThrSerLysAsnGluLeu                              290295300                                                                     PheLeuAsnGluLeuAsnThrMetProGlyPheThrAspPheSerMet                              305310315320                                                                  TyrProLeuLeuTrpGluAsnMetGlyLeuLysTyrSerAspLeuIle                              325330335                                                                     GluGluLeuIleGlnLeuAlaLeuAsnArgPheLys                                          340345                                                                        __________________________________________________________________________

What is claimed is:
 1. A purified protein comprising the amino acidsequence of SEQ ID NO:2.
 2. A purified nucleotide sequence encoding theamino acid sequence of SEQ ID NO:2.
 3. A purified nucleotide sequencecomprising the nucleotide sequence of SEQ ID NO:1.
 4. A purifiednucleotide sequence comprising the nucleotide sequence selected from thegroup consisting of SEQ ID NO:3, SEQ ID NO:4, and SEQ ID NO:5.
 5. Avector selected from the group consisting of plasmid pAT201 deposited inthe C.N.C.M. under accession number I-1277 and plasmid pAT202 depositedin the C.N.C.M. under accession number I-1291.
 6. A purified proteincomprising an amino acid sequence,wherein the amino acid sequence isencoded by the nucleotide sequence of SEQ ID NO:1, or the amino acidsequence is encoded by a first nucleotide sequence wherein a complementof the first nucleotide sequence hybridizes to a second nucleotidesequence comprising the nucleotide sequence of SEQ ID NO:1 if thecomplement of the first nucleotide sequence and the second nucleotidesequence are held together at 65° C. overnight in a solution containing0.1%SDS, 0.7% skimmed milk powder, and 6X SSC, wherein the protein issynthesized in Gram-positive bacteria expressing a low level ofresistance to vancomycin, wherein the resistance is not inducible byteicoplanin.
 7. A purified peptide fragment of the protein of claim 6,wherein the peptide fragment comprises the amino acid sequence ofresidues 110-305 of SEQ ID NO:2.
 8. The peptide fragment of claim 7,wherein the peptide fragment is recognized by an antibody to a VanBprotein and not recognized by an antibody to a VanA or VanC protein. 9.A purified nucleotide sequence encoding the protein of claim
 6. 10. Thesequence of claim 9, wherein the sequence is RNA.
 11. A purifiednucleotide sequence complementary to the sequence of claim
 9. 12. Thesequence of claim 11, wherein the sequence is chemically orradioactively labeled.
 13. A purified DNA sequence comprising (a) a genecoding for the protein of claim 6, and (b) one or more gene control orregulatory elements, wherein (a) and (b) are operably linked.
 14. Thesequence of claim 13, wherein the gene (a) comprises the nucleotidesequence of SEQ ID NO:1.
 15. A vector comprising the sequence of claim13.
 16. A cell transformed with the sequence of claim
 9. 17. The cell ofclaim 16, wherein the cell is a Gram-positive cocci bacteria.
 18. A cellcomprising the vector of claim
 15. 19. The cell of claim 18, wherein thecell is a Gram-positive cocci bacteria.
 20. The cell of claim 18,wherein the cell is E. coli BM2973.
 21. A monoclonal or polyclonalantibody which specifically binds to the protein of claim 6, and doesnot specifically bind to the VanA protein or the VanC protein.
 22. Amonoclonal or polyclonal antibody specific for the protein fragment ofclaim
 7. 23. A kit comprising (a) the antibody of claim 22, and (b) areagent for detecting an antibody-antigen complex.
 24. The kit of claim23, wherein the antibody is chemically or radioactively labelled. 25.The kit of claim 23, further comprising one or more elements selectedfrom the group consisting of a reagent for lysing a cell, vancomycin, anantibody specific for a VanA protein, and an antibody specific for aVanC protein.
 26. The kit of claim 25, wherein the vancomycin is in anaqueous solution.
 27. A kit comprising the sequence of claim 11 and oneor more elements selected from the group consisting of the nucleotidetriphosphates dATP, dCTP, dTTP, and dGTP; a DNA polymerase; and anucleotide probe specific for a VanA gene or a VanC gene.
 28. A methodcomprising the steps of:(a) contacting a sample comprising a DNAsequence with the sequence of claim 11; (b) amplifying the DNA sequenceto obtain a detectable quantity of amplified DNA; and (c) detecting thedetectable quantity of amplified DNA.
 29. The method of claim 28,wherein the contacting step is hybridizing in the presence of a DNApolymerase and the nucleoside triphosphate dATP, dCTP, dTTP, and dGTP.30. The method of claim 28, wherein the amplifying step is thepolymerase chain reaction (PCR).
 31. A method comprising the stepsof:(a) contacting a sample comprising a DNA sequence with the sequenceof claim 11, a nucleotide fragment of a VanA gene, and a nucleotidefragment of a VanB gene, wherein the nucleotide fragment of the VanAgene and the nucleotide fragment of the VanB gene each comprise morethan one nucleotide; (b) amplifying the DNA sequence to obtain adetectable quantity of amplified DNA; and (c) detecting the detectablequantity of amplified DNA.
 32. The method of claim 31, wherein theamplifying step is the polymerase chain reaction (PCR).